Cyanine dyes have been widely used for labeling biomolecules for a variety of applications such as DNA sequencing. See, for example, U.S. Pat. No. 5,571,388, incorporated herein by reference, for exemplary methods of identifying strands of DNA using cyanine dyes. Scientists favor using cyanine dyes in biological applications because, among other reasons, many of these dyes operate in the near IR (NIR) region of the spectrum (600-1000 nm). This makes these cyanine dyes less susceptible to interference from autofluorescence of biomolecules.
Other advantages of cyanine dyes Include: 1) cyanine dyes strongly absorb and fluoresce light; 2) many cyanine dyes do not rapidly bleach under the fluorescence microscope; 3) cyanine dye derivatives can be made that are simple and effective coupling reagents; 4) many structures and synthetic procedures are available and the class of dyes is versatile; and 5) cyanine dyes are relatively small (a typical molecular weight is about 1,000 daltons) so they do not cause appreciable steric interference in a way that might reduce the ability of a labeled biomolecule to reach its binding sight or carry out its function.
Despite their advantages, many of the known cyanine dyes have a number of disadvantages. Some known cyanine dyes are not stable in the presence of certain reagents that are commonly found in bioassays. Such reagents include ammonium hydroxide, dithiothreitol (DTT), primary and secondary amines, and ammonium persulfate (APS). Further, some known cyanine dyes lack the thermal and photostability that is necessary for biological applications such as DNA sequencing and genotyping.
For these reasons, improved, stable cyanine dyes are needed, especially for use in labeling biomolecules.